The Plant Journal (2002) 31(2), 149±160
Searching limiting steps in the expression of chloroplastencoded proteins: relations between gene copy number,
transcription, transcript abundance and translation rate in
the chloroplast of Chlamydomonas reinhardtii
Stephan Eberhard, Dominique Drapier and Francis-Andre Wollman*
Unite propre de recherche CNRS 1261, 13 rue Pierre et Marie Curie, F-75005 Paris, France
Received 3 December 2001; revised 8 March 2002; accepted 22 March 2002.
*
For correspondence (Fax 33 15841 5022; e-mail: [email protected]).
Summary
We performed a systematic investigation of the quantitative relationship between genome copy number,
transcription, transcript abundance and synthesis of photosynthetic proteins in the chloroplast of the
green algae Chlamydomonas reinhardtii grown either in mixotrophic or phototrophic conditions. The
chloroplast gene copy number is lower in the latter condition and the half-life and accumulation levels of
most chloroplast transcripts are signi®cantly reduced, although the relative rates of protein synthesis
remain similar. Our study shows that, in most instances, chloroplast protein synthesis is poorly sensitive
to changes in gene copy number or transcript abundance in the chloroplast. Treatment with 5-¯uoro-2¢deoxyuridine, that inhibits chloroplast DNA replication and decreases extensively the number of copies
of the chloroplast genome, had limited effects on the abundance of most chloroplast transcripts and
little if any effect on the rates of protein synthesis. When using rifampicin, that selectively inhibits
chloroplast transcription, we found no direct correlation between the level of transcripts remaining in
the chloroplast and the rates of chloroplast protein synthesis. For two chloroplast genes, a 90% decrease
in the amount of transcript did not cause a drop in the rate of synthesis of the corresponding protein
product. Overall, our results demonstrate that there is no gene dosage effect in the chloroplast and that
transcript abundance is not limiting in the expression of chloroplast-encoded protein.
Keywords: chloroplast, gene expression, rifampicin, FdUrd, Chlamydomonas.
Introduction
Transcriptional regulation of gene expression is widespread
in the prokaryote kingdom, mainly because translation
essentially occurs co-transcriptionally. As chloroplasts
derive from ancestral cyanobacteria, but have evolved as
endosymbiotic organelles, it is of interest to investigate the
different regulatory mechanisms that underlie the expression of the chloroplast genome. In higher plants, it has been
shown that regulation of chloroplast transcription is important with respect to the response of chloroplast gene
expression to light changes and in the various developmental phases leading from proplastids to differentiated
chloroplasts (Allison, 2000; Kuhlemeier, 1992; Mullet, 1993;
Stern et al., 1997). In C. reinhardtii, transcriptional activity of
chloroplast genes is modulated by a circadian rhythm, and
ã 2002 Blackwell Science Ltd
may to some extent be under nuclear control (Kawazoe
et al., 2000; Leu et al., 1990). However, there is now overwhelming evidence for the predominance of post-transcriptional control in chloroplast gene expression that
encompasses transcript maturation, stabilization and/or
translational activation, these steps being controlled by
general Ð as well as by target-speci®c Ð nucleus-encoded
factors (for recent reviews see Barkan and GoldschmidtClermont, 2000; Hauser et al., 1998; Rochaix, 2001; Stern and
Drager, 1998; Wollman et al., 1999; Zerges, 2000). Taken
together, the studies on chloroplast gene expression have
not yet de®ned the extent to which rates of transcription and
transcript abundance actually control protein synthesis in
the chloroplast. Furthermore, the possible in¯uence of
149
150
Stephan Eberhard et al.
synthesis rates in the chloroplast of the green algae
C. reinhardtii. To this end we combined approaches similar to those of Goodenough (1971) and Hosler et al. (1989)
who attempted either to inhibit transcription or to modify
chloroplast gene copy number. We show that despite
contrasted effects of changes in gene copy number on the
accumulation of some chloroplast transcripts, chloroplast
protein synthesis is mostly insensitive to a decrease in
gene copy number and that there is no close correlation
between transcript abundance and translation rate. Based
on recent studies (Heifetz et al., 2000), we also wished to
take into account a possible in¯uence of carbon metabolism on the pattern of chloroplast gene expression.
Therefore, both sets of experiments were undertaken
with algae grown either in mixotrophic or in phototrophic
conditions. These differential growth conditions re¯ect (i)
ideal growth conditions commonly used for photosynthetic mutants, i.e. acetate containing medium and lowlight (4 mE m±2 sec±1) and (ii) truly phototrophic conditions
for strains that are not impaired in photosynthesis, i.e.
minimum medium with bubbling of 5% CO2(g) under
higher light intensity (20 mE m±2 sec±1).
Figure 1. Panel a: chloroplast genome content (assayed by southern-blot
experiments).
Q = relative chloroplast genome content in the indicated growth
conditions. Signals were normalized to the signal obtained with the
nuclear probe Cblp2.
Panel b: overall mRNA accumulation levels (assayed by northern-blot
experiments) in cells grown either in mixotrophic (left panel) or
phototrophic (right panel) conditions.
R = ratio of the mRNA accumulation levels in phototrophic conditions to
the levels obtained in mixotrophic conditions. Note that the lane
corresponding to phototrophic conditions is overloaded as shown by the
signal obtained for Cblp2.
carbon metabolism on the expression of the chloroplast
genome has not yet been systematically investigated.
The organelle genome is highly polyploid. When the
unicellular green alga C. reinhardtii is grown heterotrophically, the unique chloroplast contains about 80 copies of a
circular chromosome (Lau et al., 2000; Rochaix, 1995),
whereas in higher plants, a single plastid contains from 20
to 300 genome copies (Mullet, 1993; for a review see
Sugiura, 1995). In addition, a single plant cell can accommodate up to 100 plastids. Thus, there is an important
unbalance in gene copy number between the highly
polyploid chloroplasts and the nucleus of a plant cell.
This raises intriguing questions about gene dosage for
protein expression in the chloroplast, given the composition of the major chloroplast protein complexes, whose
nuclear and chloroplast-encoded subunits are present, in
most cases, in a 1±1 stoichiometry. In this study we
examined the relation between chloroplast genome copy
number, transcription, transcript abundance and protein
Results
Chloroplast gene copy number and transcript
accumulation levels for cells grown under mixotrophic or
phototrophic conditions
To assess the contribution of gene copy number and
transcript abundance to the expression level of chloroplast
proteins, we ®rst compared these ®gures in the two
growth conditions widely used for C. reinhardtii cells, i.e.
in strictly phototrophic and in mixotrophic conditions.
DNA was extracted from the two types of cell cultures and
analysed by DNA-®lter hybridization experiments. The
results obtained for the atpB gene, taken as a marker of
the content in chloroplast DNA, were quanti®ed and
normalized to the signal obtained for the nuclear gene
Cblp2 (Figure 1a). Cells grown under mixotrophic conditions displayed a chloroplast genome content twice as
high as that of cells grown in phototrophic growth
conditions, in agreement with the report of Lau et al.
(2000). Transcript accumulation levels were monitored by
RNA-®lter hybridization experiments and are shown in
Figure 1b. The signals obtained for various chloroplast
transcripts were quanti®ed and normalized to the signal
for the nuclear Cblp2 transcript. For psbD, petA, petD, psaA
and psaB, mRNA accumulation levels were reduced about
three times in cells grown in phototrophic growth conditions when compared to cells grown in mixotrophic
growth conditions. The atpA and atpB transcripts were
more affected, their accumulation levels being about 10
times lower in phototrophic conditions. In contrast, psbA
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 149±160
Transcript/translation relationships in the chloroplast
Figure 2. Panel a: Reduction in chloroplast genome copy number after
FdUrd treatment for cells grown mixotrophically (left panel) or
phototrophically (right panel). The DNA-®lter hybridizations show the
signals obtained for the chloroplast atpB and the nuclear rbcS genes,
after 24 h or 48 h of treatment with 0.5 mM FdUrd.
Panel b: Transcript accumulation levels after FdUrd treatment. The RNA®lter hybridizations show the overall transcript accumulation levels after
24 h or 48 h of 0.5 mM FdUrd treatment for various transcripts, from cells
grown either mixotrophically (left) or phototrophically (right). The nuclear
transcript rbcS serves as a loading control.
transcripts still accumulated in phototrophic conditions to
about 50% of their level in mixotrophic conditions.
Changes in transcript abundance upon a FdUrd
treatment
The thymidine analog 5-¯uoro-2¢-deoxyuridine (FdUrd) is
known to inhibit speci®cally the chloroplast thymidilate
synthase reducing further accumulation of thymidine in
the chloroplast (Wurtz et al., 1977). Incubation of
C. reinhardtii cells with 1 mM FdUrd for a week or longer
induces mutations in the chloroplast genome (Wurtz et al.,
1979). However, treatment of cells at lower FdUrd concentrations (0,5 mM) and for shorter time periods (48 h) has
limited mutagenic effects while it still inhibits chloroplast
DNA replication. Cells thus display a drop in the number of
copies of the chloroplast chromosome after several rounds
of mitotic divisions (Hosler et al., 1989; Lau et al., 2000;
Matagne and Hermesse, 1981; Wurtz et al., 1977). Here, we
monitored the reduction in chloroplast genome copy
number after 24 h and 48 h of FdUrd treatment by DNAã Blackwell Science Ltd, The Plant Journal, (2002), 31, 149±160
151
Figure 3. Panel a: Reduction of the chloroplast gene copy number upon
FdUrd treatment assayed by DNA ®lter-hybridization. atpA: chloroplast
gene; Cblp2: nuclear gene. Reduction of the chloroplast genome copy
number was close to three in this experiment.
Panel b: RNA-pulse-labelling experiment in FdUrd treated cells. Cells
grown for 48 h in the presence (right) or absence (left) of 0.5 mM FdUrd
were pulse-labelled using thaw/freeze permeabilized cells according to
Gagne and Guertin (1992) with a-33P-UTP for 15 min in order to detect
transcription of chloroplast genes. The autoradiogram of labelled
neotranscripts hybridized to DNA restriction fragments ®xed on a nylon
membrane is shown. An overall drop in transcription yield upon FdUrd
treatment is observed. Similar results were obtained with another RNA
pulse-labelling method using toluene-permeabilized cells as described in
Guertin and Bellemare (1979) (not shown).
®lter hybridization experiments (Figure 2a). Taking the
atpB gene as a marker of the chloroplast genome, we
observed the expected drop in genome copy number upon
FdUrd treatment. As a control, we used a probe against the
rbcS and/or Cblp2 (not shown) genes whose abundances
are insensitive to FdUrd, because of their nuclear origin.
We observed variations in the extent of reduction in
chloroplast gene copy number in independent experiments relative to one growth condition, probably because
of the non-irreversible but competitive mode of inhibition
of FdUrd on chloroplast replication (Hosler et al., 1989)
and/or changes in the chloroplast content for thymidine,
that should be affected by the metabolic state of the cell.
Furthermore, reduction of chloroplast gene copy number
appeared to be more pronounced when cells were grown
mixotrophically. The decrease in chloroplast genome copy
number for cells grown phototrophically showed an
average reduction factor of 2 that is close to previous
reports by Matagne and Hermesse (1981) and Hosler et al.
(1989), whereas for cells grown mixotrophically, the
decrease in genome copy number was consistantly larger,
reaching as much as 20 in one experiment (close to the
report by Lau et al., 2000).
The rate of chloroplast transcription before and after a
48-h FdUrd treatment was then assayed by RNA-pulse
labelling experiments. To this end we used either freezethaw-permeabilized (Gagne and Guertin, 1992) or toluenepermeabilized cells (Guertin and Bellemare, 1979). Both
152
Stephan Eberhard et al.
Table 1. Chloroplast transcript accumulation after FdUrd or rifampicin treatment The table gives the mean value of the ratio of mRNA
accumulation levels after 48 h of treatment with FdUr or 6 h of treatment with rifampicin to the accumulation levels in the untreated
control. Northern blots were performed from one to ®ve times, depending on the experiment. Signals obtained for the different
chloroplast transcripts were normalized to the accumulation levels of the unaffected Cblp2 and/or rbcS transcripts, of nuclear origin
Standard deviations to the mean value of several experiments are indicated in brackets for the rifampicin experiment. As the ratio of
reduction in chloroplast gene copy number ¯uctuated between independent experiments (see text), accumulation levels shown for the
FdUrd treatment are relative to the described experiment only, and thus no standard deviation is indicated
48h FdUrd
6h rifampicin
Transcript
Mixo.
Photo.
Mixo.
Photo.
psbA
psbD
petA
petD
psaA
psaB
atpA
atpB
rbcL
0.5
0.8
0.9
0.6
0.25
0.5
0.18
0.9
1
0.5
1.2
0.4
1
0.7
0.6
0.4
0.9
0.9
0.51(+/-0.01)
0.24(+/-0.13)
0.30(+/-0.07)
0.42(+/-0.06)
0.32(+/-0.03)
0.22(+/-0.01)
0.12(+/-0.08)
0.66(+/-0.14)
0.38(+/-0.03)
0.59(+/-0.08)
0.5(+/-0.13)
0.02(+/-0.01)
0.10(+/-0.01)
0.07(+/-0.01)
0.09(+/-0.01)
0.02(+/-0.01)
0.07(+/-0.02)
0.24(+/-0.01)
Figure 4. Chloroplast protein synthesis after FdUrd treatment.
Cells were pulse-labelled in presence of cycloheximide (inhibitor of
cytoplasmic translation) with 14C-acetate for 5 min after 24 h or 48 h of
0.5 mM FdUrd treatment for cells grown either mixotrophically (left
panel) or phototrophically (right panel). An equal amount of cells was
loaded onto each lane. Correspondence between genes and proteins are
as follows: PSII complex: psbC (apo-CP43), psbB (apo-CP47), psbD (D2)
and psbA (D1). The b6f complex: petA (cyt f) and petD (SuIV). The PSI
complex: psaB (psaB). The ATP synthase complex: atpA (a) and atpB (b).
RubisCo: rbcL (LS, large subunit). The pulse-labelling experiments were
carried out on the same samples that were used for RNA extraction and
analysis.
methods gave the same qualitative information that is
illustrated in Figure 3 in the case of thaw/freeze permeabilized cells. The extent of FdUrd-induced reduction in
chloroplast gene copy number was approximately 3 in this
particular experiment (Figure 3a). It was accompanied by a
parallel decrease in the transcription rate of the four genes
that we assayed, atpA, atpB, petA and rbcL (Figure 3b).
This observation suggests that most if not all, copies of the
chloroplast chromosome are transcriptionally active in
Chlamydomonas. We then assayed the consequences of
the decreased rate of chloroplast transcription, due to the
reduction in genome copy number, on the abundance of
various chloroplast transcripts (Figure 2b and Table 1).
RNA-®lter hybridization signals obtained for nine chloroplast transcripts, representative of the mRNAs for subunits of the ®ve major photosynthetic complexes, PSII, PSI,
Cytb6f, ATP-synthase and RubisCo, were quanti®ed and
normalized to the signal obtained for the nuclear transcript
rbcS. Given the fact that the extent of reduction in
chloroplast genome content was different in independent
experiments (see above), only one experiment by growth
condition is shown, and therefore no standard deviations
are indicated for the transcript accumulation levels.
However, the same qualitative variations of the various
transcript accumulation levels were reproducibly observed
in independent experiments. Under mixotrophic growth
conditions (Figure 2b, left panel), four transcripts, psbD,
petA, atpB and rbcL, showed little if any changes after 48 h
of FdUrd treatment. Three transcripts, psaB, psbA and
petD, showed a signi®cant but limited decrease reaching
about 50% of their initial level after 48 h of treatment. Last,
two transcripts, atpA and psaA, decreased extensively
after 48 h of treatment, reaching about 20% of their initial
accumulation level. Most of the chloroplast transcripts
tested behaved similarly when the FdUrd treatment was
applied to algae grown in phototrophic conditions. For
instance the levels of psbD, atpB and rbcL transcripts
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 149±160
Transcript/translation relationships in the chloroplast
153
the large subunit of RubisCo (LS) show a signi®cant
decrease in synthesis rates after 24 h of FdUrd treatment.
The accumulation of chloroplast-encoded proteins was
not altered by the FdUrd treatment in either growth
condition, as revealed by gel electrophoresis and conventional immunodetection experiments using speci®c antibodies (not shown). Furthermore, the accumulation of the
whole set of proteins in thylakoid membranes was not
altered by the FdUrd treatment in either growth conditions
as monitored by membrane puri®cation and Coomassie or
silver staining of thylakoid proteins separated by gel
electrophoresis (not shown).
Changes in transcript abundance upon inhibition of
chloroplast transcription by rifampicin
Figure 5. Inhibition of chloroplast transcription by rifampicin.
Rifampicin was added for 6 h at 350 mg ml±1 to cells grown either
mixotrophically (upper panel) or phototrophically (lower panel), before
the labelling of neosynthesized mRNAs with 32P-a-UTP for 15 min using
toluene-permeabilized cells. The autoradiogram of labelled neotranscripts
hybridized to DNA restriction fragments ®xed on a nylon membrane is
shown.
remained poorly sensitive to the FdUrd treatment whereas
the atpA transcript decreased most in both conditions
(Figure 2b, right panel and Table 1). However, the psaA
transcript became less sensitive to FdUrd in phototrophic
conditions while the petA transcript showed an opposite
behaviour.
Rates of chloroplast protein synthesis in FdUrd treated
cells
The contrasted behaviour of distinct sets of chloroplast
transcripts upon reduction of chloroplast genome copy
number, raised the possibility that the pattern of protein
expression in the chloroplast would be extensively modi®ed after FdUrd treatment. In particular, the rates of
synthesis of the atpA and petA products, the a subunit of
CF1 of the ATP synthase complex and the cyt f of the b6f
complex, could be altered after FdUrd treatment in
mixotrophic and phototrophic conditions, respectively.
The 14C-acetate-pulse labelling experiments of chloroplast
translates, performed with FdUrd treated cells, are shown
in Figure 4. In mixotrophic conditions (Figure 4, left panel),
although some limited decrease in protein labelling
occurred, we observed no major changes in the relative
rates of chloroplast polypeptide synthesis. In particular,
the moderate drop in labelling of the a subunit of the ATP
synthase after 48 h of FdUrd treatment did not match the
drastic drop in the accumulation of atpA transcripts. In
phototrophic conditions (Figure 4, right panel), only did
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 149±160
Rifampicin selectively and irreversibly binds to the b
subunit of the E. coli RNA-polymerase, inhibiting further
transcription initiation (Campbell et al., 2001; McClure and
Cech, 1978; for a review see Richardson and Greenblatt,
1996). The same mode of action of rifampicin has been
described on the chloroplast-encoded, bacterial-like, RNApolymerase (Surzycki, 1969). Full inhibition of chloroplast
transcription can be obtained by treating cells for 1 h with
350 mg ml±1 rifampicin (Goodenough, 1971; Miller and
McMahon, 1974; Surzycki and Rochaix, 1971; Surzycki,
1969; and Guertin and Bellemare, 1979). We thus chose to
further investigate the transcript/translate relationship in
the chloroplast, using cells grown for 3 and 6 h in the
presence of 350 mg ml±1 rifampicin in either mixotrophic or
phototrophic conditions. A longer treatment with rifampicin, up to 20 h, was lethal for the cells.
We ®rst assayed the ef®ciency of rifampicin in inhibiting
chloroplast transcription by RNA-pulse-labelling experiments. In vivo transcription of ®ve chloroplast genes was
readily detected in the untreated controls using toluene
permeabilized cells (Guertin and Bellemare, 1979),
whereas no signal was obtained for atpA, petA, psbA
and rbcL when cells were treated with rifampicin for 6 h
(Figure 5). Therefore, chloroplast transcription of these
genes was fully inhibited in our experimental conditions,
which indicates that they are transcribed by a rifampicinsensitive RNA-polymerase of bacterial origin (PEP, or
Plastid Encoded Polymerase). Only did the atpB gene
show some residual transcription under mixotrophic
growth conditions (Figure 5, upper panel): a faint band
could still be observed for atpB after 6 h of rifampicin
treatment (< 1% of the control). This could be indicative of
the presence of a rifampicin-insensitive nuclear-encoded
polymerase (NEP) similar to the one which has been
described in studies with higher plant chloroplasts
(Hajdukiewicz et al., 1997; Hedtke et al., 1997). However,
this signal was not detected when the RNA pulse-labelling
experiment was performed on cells grown under photo-
154
Stephan Eberhard et al.
Figure 6. Transcript accumulation levels after rifampicin treatment.
Cells were grown either under mixotrophic (left) or phototrophic (right)
conditions in the presence of 350 mg ml±1 of rifampicin for 3±6 h, and
transcript accumulation analysed by RNA-®lter hybridizations. The
nuclear transcript Cb lp2 served as a loading control.
trophic conditions (Figure 5, lower panel). The experimental procedure used to assay in vivo transcription of
chloroplast genes has previously been assumed to allow
detection of chloroplast, but not nuclear, transcription
(Guertin and Bellemare, 1979; Kawazoe et al., 2000). In
agreement with this view, our attempts to detect transcription of the nuclear genes rbcS and Cblp2 did not give
rise to any signal with this method (not shown).
We then addressed the stability of the transcripts in the
chloroplast by performing RNA-®lter hybridization experiments (Figure 6). Given that the generation time of
C. reinhardtii in these asynchronous cultures is 5 h in
phototrophic conditions and 12 h in mixotrophic conditions, cells should divide at most once or twice during the
duration of the rifampicin treatment. Therefore, assuming
that rifampicin completely blocks transcription soon after
addition, the level of the most long-lived transcripts should
decrease by a factor of 2 (for mixotrophic conditions) and 4
(for phototrophic conditions), due to their dilution during
the experiment. Transcript accumulation levels of various
chloroplast gene were analysed by RNA-®lter hybridization
experiments, quanti®ed and normalized to the signal
obtained for Cblp2 or rbcS (not shown), two transcripts
that are unaffected by the rifampicin treatment because of
their nuclear origin. One must note that, on Figure 6,
accumulation levels can not be compared between the two
growth conditions, because experiments were realised on
different days using different transfers of RNA to nylon
membranes and differently labelled probes. When grown
in mixotrophic conditions (Figure 6, left panel) a majority
of chloroplast transcripts from rifampicin-treated algae
behaved in the same way. Their accumulation level was
Figure 7. Chloroplast protein synthesis after rifampicin treatment.
Upon addition of 350 mg ml±1 rifampicin for 3±6 h, cells were subjected to
a 5 min 14C-acetate protein pulse-labelling experiment in the presence of
cycloheximide. Labelled neosynthesized proteins were separated by gel
electrophoresis and proteins transferred to a PVDF membrane.
Radioactivity was revealed using a PhosphorImager (panel A). Loading
was controlled by directly blotting the PVDF membrane with an antibody
against CGE1, a nucleus-encoded protein (panel B). Correspondence
between genes and proteins are as indicated for Figure 5. The pulselabelling experiments were performed on the same samples that were
used for RNA extraction and analysis.
about 30% to 40% of that in untreated cells (Table 1). Two
transcripts showed contrasting behaviours: the atpA transcript decreased more severely, reaching about 10% of its
original level, whereas the atpB transcript was poorly
sensitive to rifampicin, still showing 70% of its original
accumulation level after 6 h of treatment (Table 1).
As a general rule, cells treated with rifampicin in
phototrophic conditions displayed a more drastic decrease
in their chloroplast mRNA content than their mixotrophic
counterparts. The majority of the transcripts dropped
below 10% of their original level after 6 h of rifampicin
treatment, with atpA and petA being the most affected
(Figure 6, right panel and Table 1). Noticeably, the atpB
transcript, whose accumulation level was poorly sensitive
to the rifampicin treatment in mixotrophic growth conditions, was highly affected in phototrophic conditions. In
contrast, the psbA, psbD and rbcL transcripts showed a
comparable behaviour in both growth conditions
(Table 1).
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 149±160
Transcript/translation relationships in the chloroplast
Chloroplast protein synthesis in cells treated with
rifampicin
Under mixotrophic conditions, the rates of synthesis of
most chloroplast-encoded proteins did not decrease even
after 6 h of treatment with rifampicin, despite the signi®cant changes in transcript abundance that we observed in
several instances (Figure 7a, left panel). Here again, the
rate of synthesis of the a subunit of the chloroplast
ATPsynthase showed no signi®cant changes whereas the
corresponding atpA transcript dropped dramatically after
rifampicin treatment. The synthesis of cytochrome f even
seemed to increase slightly, whereas the accumulation
level of the corresponding petA transcript was much
reduced after 6 h of rifampicin treatment. We noted that
the synthesis of the PSII subunit D1, but not of D2 (another
subunit of PSII) decreased whereas the transcripts levels of
the two subunits were similarly affected. For cells grown
under phototrophic conditions, the dramatic drop in the
level of most chloroplast transcripts was not accompanied
by reduced rates of synthesis for most of the chloroplast
proteins. However, the pattern of protein labelling varied
markedly with the time of rifampicin treatment (Figure 7a,
right panel). LS showed a continuous and marked
decrease in its rate of synthesis after rifampicin treatment.
The synthesis of many polypeptides, among which the
ATP-synthase subunits a and b, cyt f and suIV from the
cytochrome b6f complex, appeared to be transiently upregulated (compare the 0 and 3 h lanes on the right panel
of Figure 7), then returning to either the initial level as for
the a subunit and cytochrome f, or even severely decreasing as for the b subunit. Figure 7(b) shows direct
immunoblotting of the membrane with an antibody
against the nucleus-encoded Chloroplast GrpE homologue
1 (CGE1) protein (Schroda et al., 2001) that was used as a
loading control. The result shows that the 3 and 6 h lanes
are slightly overloaded, when compared to the untreated
control, but this can not on its own account for the upregulation of synthesis of most chloroplast translates at
the 3 h point. Furthermore, the up-regulation of synthesis
after 3 h of rifampicin treatment in phototrophic conditions was reproducibly observed in three independent
experiments. The major PSII subunits, apoCP47/apoCP43/
D2 and particularly D1 showed a marked and continuous
increase in synthesis after 6 h of rifampicin treatment.
These changes in the rates of synthesis of the chloroplast±encoded products were not accompanied by any
signi®cant changes in their accumulation levels, as
checked by immunoblotting with speci®c antibodies (not
shown). Also, accumulation of thylakoid membrane
proteins was not altered by rifampicin treatment in either
growth conditions as monitored by membrane puri®cation
and Coomassie or silver staining of thylakoid proteins
separated by gel electrophoresis (not shown).
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 149±160
155
Discussion
A common feature of bacteria is that transcription rate and
mRNA abundance most often control the rates of protein
synthesis. Since chloroplasts derive from ancestral cyanobacteria, our aim was to provide a better view of the pretranslational contributions to the expression of chloroplast-encoded proteins. The use of FdUrd and rifampicin in
two distinct growth conditions allowed us to demonstrate
that extensive changes in genome copy number, in
transcription rates and in the steady-state level of chloroplast transcripts are not directly correlated to changes in
the rate of synthesis of most chloroplast-encoded proteins.
Transcription of chloroplast genes
Using FdUrd as an inhibitor of chloroplast DNA replication,
we observed a parallel decrease in genome copy number
and transcription rates in the chloroplast. This supports
the view that most copies of the chloroplast chromosome
are transcriptionally active and that the RNA polymerases
are not present in limiting concentration. In most instances
however, the decreased rates of transcription due to lower
chloroplast ploidy, were not accompanied by signi®cant
changes in the abundance of the vast majority of the
chloroplast transcripts tested. Among the exceptions were
the decreased accumulation of psaA transcripts for algae
grown in mixotrophic conditions, of petA transcripts for
algae grown phototrophically and atpA transcripts in
either growth condition. The poor sensitivity of the posttranscriptional step to FdUrd treatment can be readily
explained by the presence of limiting amounts of nucleusencoded factors that select a subset of neosynthesized
transcripts in the chloroplast, protecting them from
nucleolytic degradation after transcription. Therefore, the
concentration of these factors (and neither the genome
copy number nor the actual transcription rate) would
determine the abundance of transcripts in the chloroplast.
Nuclear factors that protect chloroplast mRNAs from
nucleolytic degradation by acting on their 5¢UTR have
been described for most of the chloroplast transcripts that
we tested but not for atpA (see Barkan and GoldschmidtClermont, 2000; Nickelsen, 1998; Stern and Drager, 1998;
Wollman et al., 1999; Monde et al., 2000). If such a factor is
missing for atpA, its accumulation would be predicted to
follow the fraction of transcriptionally active chloroplast
chromosomes contrarily to the other transcripts that can
accumulate in the chloroplast in proportion to the concentration of their nuclear-encoded stabilizing factors. An
additional prediction of this proposal is that the atpA
mRNAs should decline more severely than all other
chloroplast transcripts when transcription is blocked, a
behaviour that we indeed observed after treatment of the
algae with rifampicin.
156
Stephan Eberhard et al.
Relation between transcript accumulation levels and
rates of translation
A correlation between transcript accumulation levels and
protein synthesis rates has been established in some
instances in higher plants (Mullet, 1993; Pfannschmidt
et al., 1999; Rapp et al., 1992). However, other developmental studies have shown that transcriptional activity is
not necessarily tightly linked to rates of chloroplast protein
synthesis, pointing to an extensive post-transcriptional
control of chloroplast gene expression (Mullet, 1988). The
analysis of several translation-defective strains of
C. reinhardtii has shown that the level of the non-translated transcript either increased, as for atpA (Drapier et al.,
1992), or decreased, as for psbA (Girard-Bascou et al.,
1992) or petA (Wostrikoff et al., 2001). Thus, as discussed
by Nickelsen (1998), there is no general rule linking
translation and mRNA stability in the chloroplast of
C. reinhardtii.
In the present study we found only in one instance, atpB,
some correlation between transcript abundance and translation rates. In the experiments where the accumulation of
atpB transcripts was mostly unaffected (namely after
FdUrd treatment performed in either growth condition or
rifampicin treatment of algae grown in mixotrophic conditions) the rates of synthesis of the b subunit was not
affected. However, in the single experiment where the
accumulation level of the atpB transcript was dramatically
reduced (rifampicin treatment of algae grown in phototrophic conditions), the rate of synthesis of the b subunit
displayed a signi®cant decrease. In all other cases, our
study provides extensive experimental support to the lack
of a direct relationship between translation rates and
transcript abundance in C. reinhardtii chloroplasts. The
atpA and petA transcripts that decreased severely after
FdUrd or rifampicin treatment still allowed rates of protein
synthesis that were comparable to those observed in the
untreated control. For instance, in rifampicin-treated cells
grown phototrophically, as little as 2% of atpA and petA
transcripts were suf®cient to provide near wild-type synthesis of the corresponding proteins. The fact that severely
reduced amounts of those transcripts still permits near
wild-type synthesis of the corresponding proteins may be
explained by either of two ways: (i) a smaller amount of
transcripts is more intensively translated or (ii) in the
regular growth conditions with no drug added, only a
subset of those transcripts is actually loaded onto polysomes for translation. That the concentration of chloroplast transcripts is not rate-limiting for translation is
consistent with a previous report by Hosler et al. (1989)
that a reduction in chloroplast genome copy number in
C. reinhardtii cells grown phototrophically produced a
decrease in the accumulation levels of the chloroplast
atpA, rpl2 and rbcL transcripts, whereas synthesis of the
corresponding proteins, the a subunit of ATP-synthase,
the r-protein L1 and LS remained largely unaffected
(however, the behaviour of LS reported by Hosler et al.
(1989) is in contrast with our data: we observed a reduction
in synthesis rate of LS for the FdUrd treatment performed
in phototrophic conditions). Overall, it appears that translation must be controlled by rate-limiting nucleus-encoded
factors imported in the chloroplast (for reviews see Hauser
et al., 1998 and Zerges, 2000), as has been documented for
pet494p (Steele et al., 1996) or pet111p (Green-Willms
et al., 2001), which are nuclear-encoded translational
activators of the COX3 and COX2 mRNA in yeast
mitochondria, respectively. Thus, as pointed out by
Hosler et al. (1989), there may be two distinct pools of
mRNA in the chloroplast, a pool of non-translatable
mRNAs and a pool of mRNAs that are activated for
translation by nucleus-encoded factors.
A remarkable illustration of the prominent role of
translational regulation can be found in our study of the
expression of the psbA and rbcL genes, which encode,
respectively, the PSII subunit D1 and LS: a similar decrease
in psbA transcripts in mixotrophically grown and phototrophically grown algae (for both the FdUrd and rifampicin
experiments) was accompanied in one case by a down
regulation in the synthesis of the D1 protein, whereas
it was up-regulated in the other case. This behaviour
re¯ects the complex translational regulation for this
PSII subunit that encompasses ribosome pausing
(Kim et al., 1991; Zhang et al., 2000) and is the most
sensitive to photoinduced damage (Danon and May®eld,
1994a, 1994b; Kim and May®eld, 1997; Trebitsch et al.,
2000). Up-regulation of the synthesis of the D1 subunit
in high light without signi®cant changes in psbA transcript
accumulation levels has also been reported by Shapira
et al. (1997). For rbcL, comparable accumulation levels
of the transcript in cells treated with FdUrd in either
growth conditions, led to a decrease in the synthesis rate
of the corresponding LS protein for cells grown phototrophically, whereas it remained constant for cells grown
mixotrophically.
Effect of growth conditions
Under standard conditions, our results are in agreement
with those published by Lau et al. (2000), i.e. that
C. reinhardtii cells grown mixotrophically contain about
twice as much chloroplast DNA as cells grown phototrophically. A likely explanation, given the shorter generation time of cells grown in phototrophic conditions (5 h)
than in mixotrophic growth conditions (12 h), is that
higher number of mitotic cell divisions per time unit lead
to an increased partitioning of non-replicated chloroplast
genomes, as is the case for bacteria. The lower decrease in
chloroplast genome copy number upon FdUrd treatment
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 149±160
Transcript/translation relationships in the chloroplast
of cells grown phototrophically may also originate from
their lower polyploidy, if one assumes that there is a lower
limit for the number of chloroplast chromosomes required
for cell viability.
Photosynthetic activity and light quality are also known
to participate in the regulation of chloroplast genome
expression. At the transcriptional level, it has been shown
that psbD, psbC and petG genes are under the control of a
blue-light activated promoter in higher plant chloroplasts
(Christopher et al., 1992; for a review see Stern et al., 1997).
In C. reinhardtii, variations in chloroplast transcription
rates were observed in a circadian way, with a peak of
transcription at the onset of the light period (Leu et al.,
1990). These results have been extended to a general lightdependent variation in chloroplast mRNA levels, that are
due to both transcriptional and post-transcriptional regulations, the degradation of chloroplast transcripts being up
to ®ve times higher in the light period when compared to
the dark period of synchronously grown cultures (Salvador
et al., 1993). We observed a similar change in chloroplast
mRNA stability when comparing the rifampicin-treated
algae grown under mixotrophic or phototrophic conditions. Only did the rbcL transcripts show limited destabilization in phototrophic conditions and the two PSII
transcripts tested (psbA and psbD) remained as stable as
in mixotrophic conditions. All the other mRNAs tested
displayed a severe drop in transcript stability, their
degradation being four to ®ve times higher in phototrophic
conditions. The higher stability of the psbA transcript
correlates well with the limited changes in its abundance
when changing growth conditions (Figure 1b). The comparable stability of the rbcL transcript in both conditions is
in good agreement with a report by Shiina et al. (1998)
showing that in tobacco, accumulation levels of rbcL
transcripts are independent of light, although in their
case modi®cation of transcription rate and mRNA stability
appeared to play a role. On the other hand, this signi®cantly lower life-time of most chloroplast transcripts for
cells grown phototrophically fully accounts for their lower
steady-state mRNA levels, when compared to cells grown
mixotrophically, non-regarding the limited difference in
gene copy number (by only a factor of 2). Particularly, the
very short half-life of atpA and atpB transcripts in
phototrophic growth conditions (Figure 6, right panel)
may explain on its own the 10 times drop in their steady
state accumulation levels when compared to cells grown
mixotrophically (Figure 1b).
The decreased half-life of atpB and psaB in phototrophic
conditions may be due to the same degradation process
that is triggered by DTT for these two transcripts (Salvador
and Klein, 1999), suggesting a role of thioredoxin mediated regulations of chloroplast transcript stability. Further
support for a regulatory role of thioredoxins in phototrophic conditions comes from the examination of the
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 149±160
157
pattern of synthesis of chloroplast-encoded proteins in
phototrophically grown algae treated with rifampicin for
6 h (Figure 7, right panel): it became enriched in its PSII
components. Several reports suggest that translation of
the psbA transcript is strongly enhanced under phototrophic growth conditions due to a translational activation
complex that preferentially binds the psbA messenger in
the light through a thioredoxin-controlled mechanism
(Danon and May®eld, 1994a, >1994b; Fong et al., 2000;
Trebitsch et al., 2000). It thus appears that photosynthetic
activity has a major impact on chloroplast gene expression, most likely through the action of redox activated
regulatory factors.
Overall, the fact that chloroplast protein synthesis rates
are mostly insensitive to mRNA accumulation levels
allows the algae to respond to physiological changes in
chloroplast genome copy number and transcript accumulation levels that naturally take place, when the growth
conditions vary with naturally occurring changes in availability in carbon sources. Post-transcriptional regulatory
mechanisms therefore offer a good preservation of
photosynthetic competence in changing environmental
conditions, although gene copy number and mRNA levels
in the chloroplast would be affected.
The lack of correlation between gene copy number,
transcript abundance and protein translation in the chloroplast illustrate the deep evolution in gene expression that
has developed with the engulfment of a prokaryote in a host
cell. The control of chloroplast protein expression by
nuclear-encoded factors provides the framework for our
current understanding of these speci®c traits of organellar
gene expression. How these nuclear-encoded factors are
delivered to their targets in the organelle therefore stands
as a key issue for further studies of chloroplast biogenesis.
Experimental procedures
Strains and growth conditions
The C. reinhardtii wild-type (A1, mt-) strain, a derivative of the
137c strain presented in Harris (1989) was used for all the
described experiments. For mixotrophic growth conditions, cells
were grown in a liquid Tris-Acetate-Phosphate (TAP) medium
(pH 7.2), as described in Harris (1989), under continuous low light
(4 mE m±2 s±1) at 25°C and agitation. For phototrophic growth
conditions, cells were grown in a minimum medium (pH 7.2)
(comparable to the HS medium described in Harris (1989)) under
an illumination of 20 mE m±2 s±1 and with the addition of 5%
bubbled CO2(g) at 25°C and agitation.
Treatment with drugs: FdUrd treatment
Cells were incubated for up to 48 h with 0.5 mM of 5-¯uoro-2¢deoxyuridine (Sigma), a treatment known to reduce chloroplast
genome copy number (Wurtz et al., 1977).
158
Stephan Eberhard et al.
Table 2. Intragenic DNA fragments from chloroplast or nucleus origin, used for hybridization of Northern or Southern blots.
Gene
psbA
psbD
petA
petD
psaA
psaB
atpA
atpB
rbcL
rbcS
Cblp2
Origin
entire R14 fragment of the chloroplast DNA
entire R3 fragment of the chloroplast DNA
HindIII fragment of the chloroplast R26 fragment
HindIII fragment of the chloroplast R26 fragment
HindIII-EcoRI fragment of the chloroplast R11 fragment
Bam01 fragment of the chloroplast DNA
EcoRI-PstI fragment of the chloroplast R7 fragment
EcoRI-KpnI fragment of the chloroplast Ba5 fragment
HindIII fragment of the chloroplast R15 fragment
AlwNI-SstII fragment of the coding region of rbcS2
entire cDNA of the nuclear Cblp2 gene
Rifampicin treatment
Cells were incubated for up to 6 h with 350 mg ml±1 of rifampicin
(Sigma) to inhibit transcription of chloroplast but not nuclear
genes (Surzycki, 1969).
RNA analysis
Total RNA was extracted and analysed by Northern blots as in
Drapier et al. (1998). The bands shown in Figures 1,2 and 6
correspond only to the mature mRNAs of the corresponding
genes (in some cases, maturation intermediaries were observed,
but were discarded for quanti®cations and not included in the
®gures). Radiolabelled gene-speci®c probes were derived from
intragenic DNA fragments of nuclear or chloroplast origin
(Table 2). Labelling was performed for each probe using the
Nonaprimer Labelling Kit (Quantum, Appligene) with 5 ng of DNA
and 25 mCi of a-33P- dATP.
In vivo RNA-pulse labelling
Cells were assayed for de novo chloroplast RNA synthesis using
toluene permeabilized cells as described in Guertin and Bellemare
(1979) with the following modi®cations: cells were solubilized
with 1% toluene and pulse-radiolabelled with 165 mCi ml±1 of
a-32P-UTP (400 Ci mmol±1, Amersham) for 15 min RNA, extracted
as described in Drapier et al. (1998), was then hybridized with
unlabelled DNA restriction fragments of chloroplast or nuclear
origin separated by gel electrophoresis and transferred to a nylon
membrane. RNA pulse-labelling experiments were also performed using thaw/freeze permeabilized cells according to
Gagne and Guertin (1992) with the modi®cations described in
Sakamoto et al. (1993).
DNA-®lter hybridizations
Total DNA was prepared as described in Rochaix (1980), separated by gel electrophoresis, transferred to nylon membranes and
probed with radiolabelled intragenic DNA fragments from
chloroplast or nuclear origin.
Protein analysis
Pulse-labelling experiments were carried out as described in
Drapier et al. (1992) with 5 mCi ml±1 of 14C-acetate (50 mCi mmol±1,
Amersham) in the presence of an inhibitor of cytoplasmic
Size
2.3 kB
2.7 kB
3.5 kB
1.1 kB
2.2 kB
580 bp
947 bp
2.9 kB
890 bp
370 bp
1 kB
Reference
Rochaix (1978)
Rochaix (1978)
BuÈschlen et al. (1991)
BuÈschlen et al. (1991)
Choquet et al. (1988)
Rochaix (1978)
Rochaix (1978)
Rochaix (1978)
Rochaix (1978)
Schroda et al. (1999)
Schroda et al. (2001)
translation (6,6 mg ml±1 cycloheximide, Sigma). Proteins of
solubilized cells were separated in urea/SDS-polyacrylamide
gels as described in Piccioni et al. (1981) and transferred to a
PVDF membrane which was then exposed in a PhosphorImager
to detect radiolabelled proteins. Loading of the gels was controlled by immunoblotting of the PVDF membrane with antibodies directed against nucleus-encoded proteins (CGE1
(Schroda et al., 2001), OEE2 and/or OEE3).
Acknowledgements
The authors wish to thank Michael Schroda, Julien Fey and Yves
Choquet for stimulating discussions and/or critical reading of the
manuscript. The authors also gratefully acknowledge technical
assistance of Laetitia Martin for some of the experiments
described. Stephan Eberhard is a recipient of a fellowship from
the MinisteÁre de l'Education Nationale. This work was supported
by the UPR1261 of the Centre National de la Recherche
Scienti®que.
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